The present invention relates to the propagation of human hepatocytes in the livers of non-human animals that have been tolerized to the human cells. Such animals provide an in vivo model system of the human liver that may be used in toxicology assays and in the study of human liver diseases, including the various forms of hepatitis (in particular hepatitis B and C) and alcohol-induced liver degeneration. They may also be used as a source of human hepatocytes for reconstitution of liver tissue, thereby providing an alternative to liver transplantation.
To accurately study the physiology of human liver cells (hepatocytes), scientists need a model system in which the hepatocytes exist as they would in the intact liver. Such systems have proven to be difficult to achieve, because when hepatocytes are removed from their native environment, they tend to lose their specialized functions, or xe2x80x9cde-differentiatexe2x80x9d. The loss of liver-specific functions makes it difficult or impossible to study the normal functions of hepatocytes as well as their response to chemical or biological agents. For example, research directed toward infectious diseases of the liver, in particular viral hepatitis, has been hampered by the lack of an adequate model system. Hepatitis B and hepatitis C, and the problems that have been encountered by scientists studying these infectious and dangerous viruses, are discussed in the following subsections.
In addition, a system for propagating human hepatocytes could be used to provide cells that could be used as an alternative or adjunct to liver transplant. Currently, patients suffering from liver disease may have to wait for long periods of time before a suitable organ for transplant becomes available. After transplant, patients need to be treated with immunosuppressive agents for the duration of their lives in order to avoid rejection of the donor""s liver. A method for propagating the patient""s own cells could provide a source of functional liver tissue which would not require immunosuppression to remain viable.
Hepatitis B virus (xe2x80x9cHBVxe2x80x9d) is the prototype of the Hepadnaviridae, characterized by a unique genome structure comprising partially double-stranded DNA (Fields Virology, 1996, Third Edition, Fields, et al. eds., Lippincott-Raven, New York, pp. 2741-2742). In the United States, there are about a million carriers of HBV, and the number of carriers in the world exceeds 350 million (Fields Virology, p. 2741; Petersen et al., 1998, Proc. Natl. Acad. Sci. U.S.A. 95:310-315). In addition to causing an acute hepatitis, viral infection may lead to chronic infection and consequent liver failure and/or the development of hepatocellular carcinoma (Fields Virology, pp. 2748-2751). The development of agents that effectively treat and/or prevent the spread of the disease has been limited by the lack of good small animal model systems. Among the models recently developed are a transgenic mouse model and a xe2x80x9cTrimeraxe2x80x9d, reported in Petersen et al., 1998, Proc. Natl. Acad. Sci. U.S.A. 95:310-315 and Ilan et al., 1999, Hepatology 29:553-562, respectively.
In the transgenic mouse model of Petersen et al., a transgene encoding a hepatotoxic urokinase-type plasminogen activator was introduced into RAG-2 knockout mice, which lack mature B and T lymphocytes, and then woodchuck hepatocytes were introduced via splenic injection. The woodchuck hepatocytes replaced up to 90 percent of the mouse liver, and supported woodchuck hepatitis virus (another hepadnavirus) replication indefinitely. The replication of the virus responded to pharmacologic agents.
In the Trimera model described by Ilan et al., normal mice were preconditioned by lethal total body radiation, radioprotected with SCID mouse bone marrow cells, and then engrafted with human liver fragments infected ex vivo with hepatitis B.
Hepatitis C virus was first characterized in 1989 (Choo et al., 1989, Science 244: 359-362), but its existence had been posited for many years as an elusive entity that caused flu-like symptoms in certain patients who had received blood transfusions. Because these symptoms were sometimes followed, years later, by liver disease, the clinical syndrome was referred to as non A-non B hepatitis (xe2x80x9cNANBHxe2x80x9d).
Hepatitis C virus (xe2x80x9cHCVxe2x80x9d) is now known to be a member of the Flaviviridae family of viruses, which includes viruses that cause bovine diarrhea, hog cholera, yellow fever, and tick-borne encephalitis (Kato et al., 1990, Proc. Natl. Acad. Sci. U.S.A. 87: 9524-9528; Choo et al., 1991, Proc. Natl. Acad. Sci. U.S.A. 88: 2451-2455; Okamoto et al., 1991, J. Gen. Virol. 72: 2697-2704;Takamizawa et al., 1991, J. Virol. 65: 1105-1113). The viral genome consists of an approximately 9.5 kb single-stranded, positive-sense RNA molecule characterized by a unique open reading frame coding for a single polyprotein (reviewed in Clarke, 1997, J. Gen. Virol 78: 2397-2410 and Major and Feinstone, 1997, Hepatology 25: 1527-1538). Based upon phylogenetic analysis of the core, EI, and NS5 regions, HCV has been found to be genetically heterogeneous, with at least six genotypes and more than 30 subtypes dispersed throughout the world (Major and Feinstone, 1997, Hepatology 25: 1527-1538; Clarke, 1997, J. Genl. Virol 78: 2397-2410).
HCV has been estimated to infect 170 million people worldwide, which is more than four times the number of persons infected with human immunodeficiency virus (xe2x80x9cHIVxe2x80x9d), and the number of HCV-associated deaths may eventually overtake deaths caused by AIDS (Cohen, 1999, Science 285: 26-30). The Center for Disease Control has calculated that HCV may be harbored by 1.8 percent of the U.S. population. (Id.). The only available therapy is interferon, but most HCV isolates are resistant (Thomas et al., 1999, Hepatology 29: 1333), although more promising results were obtained when interferon was combined with ribavirin (Cohen et al., 1999, Science 285: 26-30 citing Poynard et al., 1998, Lancet 352:1426-1432 and Davis et al., 1998, N. Engl. J. Med. 339:1493-1499). Unfortunately, the interferon/ribavirin combination is less effective against the most common HCV genotype found in the U.S., with only 28 percent of persons infected with that genotype exhibiting a sustained response to treatment. (Davis et al., 1998, N. Engl. J. Med. 339:1493-1499).
The development of more successful forms of therapy (and our understanding of HCV biology) has been hampered by the absence of a good model system for HCV infection. Only humans and certain higher primates are susceptible to infection (Feinstone et al., 1981, J. Infect. Dis. 144: 588). A variety of mammalian cell systems which support the growth of HCV have been reported which rely on the use of strand-specific RT-PCR as evidence of virus replication (Major and Feinstone, 1997, Hepatology 25:1527-1538 citing Mitzutani et al., 1995, Biochem. Biophys. Res. Commun. 212: 906-911; Shimizu and Yoshikura, 1994, 68: 8406-8408; Kato et al., 1995, Biochem. Biophys. Res. Commun. 206: 863-869; Cribier et al., 1995, J. Gen. Virol. 76: 2485-2491; and Yoo et al., 1995, J. Virol. 69: 32-38).
As reviewed in Clarke (supra), there have been reports of viral replication in systems based on hepatic tissue (Ito, et al., 1996, J. Gen. Virol. 77: 1043-1054), peripheral blood mononuclear cells (Willems et al., 1996, J. Med. Virol. 42: 272-278; Zignego et al., 1992, J. Hepatology 15: 382-386), human T and B cell lines (Bertolini et al., 1993, Res. Virol 144: 281-285; Shimizu et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89: 5477-5481), human fetal liver cells (Iacovacci et al., 1993, Res. Virol. 144: 275-279), chimpanzee hepatocytes (Lanford et al., 1994, Virol. 202: 606-614), Daudi B-cells (Nakajima et al., 1996, J. Virol. 70: 3325-3329), and the human T cell leukemia virus type I-infected T cell line MT-Z (Mitzutani et al., 1995, Biochem. Biophys. Res. Comm. 212: 906-911; Sugiyama et al., 1997, J. Gen. Virol. 78: 329-336). None of these systems has, however, proved satisfactory.
Hepatitis C infected human liver tissue was transplanted into Trimera mice described in the preceding section, as reported by Galun et al., 1995, J. Infect. Dis. 172:25-30.
A newer system was recently reported by Lohmann et al. (1999, Science 285: 110-113) in which subgenomic HCV RNA replicons were transfected into a human hepatoma cell line and found to replicate to high levels. Nonetheless, this system does not generate virus and therefore is not a model of productive infection (Cohen, supra).
Reconstitution of liver tissue in a patient by the introduction of hepatocytes (also referred to as xe2x80x9chepatocyte transplantationxe2x80x9d) is a potential therapeutic option for patients with acute liver failure, either as a temporary treatment in anticipation of liver transplant or as a definitive treatment for patients with isolated metabolic deficiencies (Bumgardner et al., 1998, Transplantation 65: 53-61). Animal models have been developed for studying the effectiveness of hepatocyte transplantation in the context of pharmacologically or surgically induced liver failure (Id, citing Mito et al., 1993, Transplant Rev. 7: 35; Takeshita et al. 1993, Cell Transplant 2: 319; Sutherland et al., 1977, Surgery 82: 124; Sommer et al., 1979, Transplant Proc. 9: 578; and Demetriou et al., 1988, Hepatology 8: 1006), or for the treatment of isolated errors of metabolism (Wiederkehr et al., 1990, Transplant 50: 466; Onodera et al., 1995, Cell Transpl. 4 (Supp. 1): 541; Cobourn et al., 1987, Transpl. Proc. 19: 1002; Rozga et al., 1995, Cell Transplant 4: 237; Kay et al., 1994, Hepatology 20: 253; Matas et al., 1976, Science 192: 892; Holzman et al., 1993, Transplantation 55: 1213; Moscioni et al., 1989, Gastroenterol. 96: 1546; Groth et al., 1977, Transplant Proc. 9: 313). Use of transfected hepatocytes in gene therapy of a patient suffering from familial hypercholesterolemia has been reported in Grossman et al., 1994, Nat. Genet. 6: 335.
A major obstacle to achieving therapeutic liver reconstitution is immune rejection of transplanted hepatocytes by the host, a phenomenon referred to (where the host and donor cells are genetically and phenotypically different) as xe2x80x9callograft rejectionxe2x80x9d. Immunosuppressive agents have been only partially successful in preventing allograft rejection (Jauregui et al., 1996, Cell Transplantation 5: 353-367, citing Darby et al., 1986, Br. J. Exp. Pathol. 67: 329-339; Maganto et al., 1988, Eur. Surg. Res. 20: 248-253; Makowka et al., 1986, Transplantation 42: 537-541). The three main alternative approaches which have been explored are 1) physically shielding transplanted cells from the host immune system, for example, in an alginate-polylysine or chitosan capsule; 2) depletion of antigen presenting cells; or 3) induction of alloantigen-specific tolerance in the host (Jauregui et al., supra). Chowdhury has tested the hypothesis that intrathymic injection of donor rat splenocytes may result in suppression of allograft hepatocyte rejection in peripheral lymphocyte depleted adult rats (Jauregui et al., supra, citing Fabrega et al., 1995, Transplantation 59: 1362-1364). In that study long-term tolerization occurred with administration of splenocytes but not hepatocytes.
For successful reconstitution, the age of the donor cells has been considered significant. Cusick et al. (1997, J. Ped. Surg. 32: 357-360) report that transplanted fetal hepatocytes had a significant survival advantage over adult hepatocytes, independent of recipient age. However, Rhim et al. (1994, Science 263: 1149-1152) demonstrated that adult mouse liver cells could proliferate when introduced into the livers of congenic transgenic mice carrying a hepatotoxic transgene (urokinase under the control of the albumin promoter, which is liver-specific and only active postnatally). The donor cells were observed to have divided at least 12 times (reconstitution of an entire liver from one hepatocyte would require 28 cell doublings).
The present invention relates to the preparation of tolerized non-human animals having chimeric livers, wherein some or a majority of the hepatocytes present are human hepatocytes. It is based, at least in part, on the discovery that rats, tolerized in utero against human hepatocytes, were found to serve as long-term hosts for human hepatocytes introduced postnatally, and that the introduced hepatocytes maintained their differentiated phenotype, as evidenced by continued production of human albumin.
In a first embodiment, the present invention provides for a method of preparing a non-human animal having a liver comprising human hepatocytes, comprising (i) inducing tolerance in an immunocompetent host non-human animal, where the animal is preferably a fetus or a neonate; and (ii) introducing human hepatocytes into the tolerized animal, preferably postnatally and preferably by intra-splenic injection. In specific non-limiting embodiments, the host animal is subjected to a selection pressure which favors survival and/or proliferation of human, rather than host animal, hepatocytes.
In a second embodiment of the invention, an animal having a chimeric liver, prepared as described above, may be used as a model system for human hepatocyte function in a toxicology study. Because the human hepatocytes maintain their differentiated state and are situated in their natural anatomic location, this model system recapitulates the metabolic fate of test agents as they pass from the site of administration through the liver.
In a third embodiment of the invention, an animal having a chimeric liver may be used as a model system for human liver disease. Such model systems are particularly useful for diseases which specifically effect human (or primate), but not non-human (or non-primate) livers, such as hepatitis B and hepatitis C infection and alcohol-induced liver degeneration/fibrosis. Immunocompetent chimeric animals of the invention exhibit the further advantage of having an immune system which is intact but for exhibiting tolerance toward the human cells comprised in the animal""s liver.
In a fourth embodiment of the invention, an animal having a chimeric liver may be used as a source of human hepatocytes which may be used therapeutically. As non-limiting examples, such human hepatocytes may be used in gene therapy applications or to reconstitute liver tissue in a human host whose own liver has been substantially damaged. Large animals having a chimeric liver may be particularly desirable for such embodiments.
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